ABSTRACT Physiological aging is associated with a low grade chronic inflammatory state which has been link to numerous geriatric comorbid conditions. While there are likely multiple contributors to this ?inflammaging? phenotype, a number of recent animal model studies have implicated a breakdown in gut homeostasis as a key factor. We established that human plasma biomarkers indicative of gut epithelial barrier damage and gut microbial translocation increased with age and correlated with markers of systemic immune activation thus showing that age-related disruption of human gut homeostasis is linked to inflammaging. Prior studies have shown that the fecal microbiome is altered or ?dysbiotic? with age, with an increase in enteric commensal bacteria capable of inducing inflammation (pathobionts) and a decrease in immune regulatory bacterial metabolites (e.g. the short chain fatty acid, butyrate). Gut CD4 T cells are the largest gut mucosal T cell population and play a vital role in maintaining homeostasis by protecting against gut pathogens or translocating microbes. We demonstrated that human gut lamina propria (LP) CD4+ T cells become activated, produce inflammatory cytokines (IL-17, IFN?), and proliferate upon exposure to enteric pathobiont bacteria in vitro. We now have preliminary data demonstrating that butyrate inhibits these bacteria-driven inflammatory responses and induces expression of regulatory molecules such as PD1 and IL-10. In pilot ex vivo studies we show that older age is associated with an accumulation of IL-17-producing T helper (Th) cells (Th17), especially those that co-produce IFN?, a known inflammatory subset. Furthermore, older LP CD4 T cells expressed significantly lower levels of the co-inhibitory immune checkpoints PD1 and CTLA4 than younger LP CD4 T cells and higher levels of anti-apoptotic Bcl-2. This finding is counterintuitive as increased expression of PD1 and CTLA4 have been associated with exhaustion of peripheral blood T cells. Our overarching hypothesis is that age-associated intrinsic immune dysregulation (low co-inhibitory immune checkpoint expression) of gut CD4 T cells combined with pro- inflammatory (high pathobiont, low butyrate) gut dysbiosis will result in expansion of inflammatory Th17 cells. To explore age-associated gut CD4 T cell/microbe interactions, we propose 2 aims that will utilize an ex vivo human colon LP mononuclear cell model to gain insights into the effects of aging on microbiome-gut CD4 T cells interactions. In Aim 1, we will determine whether distinct colonic Th cells differentially express a dysregulated aging immune phenotype and identify a corresponding transcriptomics profile linked to this phenotype. In Aim 2 we will evaluate the impact of aging on immune function of colonic Th subsets in response to colonic microbiome-associated immune-stimulatory and immune-regulatory factors. These studies will provide critically needed information on the impact of age on gut CD4 T cell immunity in the setting of age-associated dysbiosis. The insights gained will support future studies in older populations designed to probe the contribution of specific pathways of gut inflammation to systemic inflammation and comorbidities.